Comprehensive DNA barcoding of the herpetofauna of Germany

We present the first comprehensive DNA barcoding study of German reptiles and amphibians representing likewise the first on the European herpetofauna. A total of 248 barcodes for all native species and subspecies in the country and a few additional taxa were obtained in the framework of the projects ‘Barcoding Fauna Bavarica’ (BFB) and ‘German Barcode of Life’ (GBOL). In contrast to many invertebrate groups, the success rate of the identification of mitochondrial lineages representing species via DNA barcode was almost 100% because no cases of Barcode Index Number (BIN) sharing were detected within German native reptiles and amphibians. However, as expected, a reliable identification of the hybridogenetic species complex in the frog genus Pelophylax was not possible. Deep conspecific lineages resulting in the identification of more than one BIN were found in Lissotriton vulgaris, Natrix natrix and the hybridogenetic Pelophylax complex. A high variety of lineages with different BINs was also found in the barcodes of wall lizards (Podarcis muralis), confirming the existence of many introduced lineages and the frequent occurrence of multiple introductions. Besides the reliable species identification of all life stages and even of tissue remains, our study highlights other potential applications of DNA barcoding concerning German amphibians and reptiles, such as the detection of allochthonous lineages, monitoring of gene flow and also noninvasive sampling via environmental DNA. DNA barcoding based on COI has now proven to be a reliable and efficient tool for studying most amphibians and reptiles as it is already for many other organism groups in zoology.

[1]  M. Kwak,et al.  DNA barcode reference data for the Korean herpetofauna and their applications , 2013, Molecular ecology resources.

[2]  Sujeevan Ratnasingham,et al.  A DNA-Based Registry for All Animal Species: The Barcode Index Number (BIN) System , 2013, PloS one.

[3]  Z. Nagy,et al.  Reliable DNA Barcoding Performance Proved for Species and Island Populations of Comoran Squamate Reptiles , 2013, PloS one.

[4]  H. Tunner.  Das Albumin und andere Bluteiweiße bei Rana ridibunda Pallas, Rana lessonae Camerano, Rana esculenta Linné und deren Hybriden , 2009 .

[5]  N. Baeshen,et al.  Biological Identifications Through DNA Barcodes , 2012 .

[6]  T. Beebee,et al.  Microsatellite analysis of natterjack toad Bufo calamita Laurenti populations: consequences of dispersal from a Pleistocene refugium , 2000 .

[7]  J. Macey,et al.  A molecular assessment of phylogenetic relationships and lineage accumulation rates within the family Salamandridae (Amphibia, Caudata). , 2006, Molecular phylogenetics and evolution.

[8]  Y. Gräser,et al.  Batrachochytrium dendrobatidis in Germany: distribution, prevalences, and prediction of high risk areas. , 2013, Diseases of aquatic organisms.

[9]  C. Corti,et al.  Mitochondrial phylogeography, contact zones and taxonomy of grass snakes (Natrix natrix, N. megalocephala) , 2013 .

[10]  P. Hebert,et al.  bold: The Barcode of Life Data System (http://www.barcodinglife.org) , 2007, Molecular ecology notes.

[11]  U. Joger,et al.  A new cryptic species of pond turtle from southern Italy, the hottest spot in the range of the genus Emys (Reptilia, Testudines, Emydidae) , 2005 .

[12]  Jeremy R. deWaard,et al.  Biological identifications through DNA barcodes , 2003, Proceedings of the Royal Society of London. Series B: Biological Sciences.

[13]  D. Tautz,et al.  Mitochondrial sequence analysis of Salamandra taxa suggests old splits of major lineages and postglacial recolonizations of Central Europe from distinct source populations of Salamandra salamandra , 2000, Molecular ecology.

[14]  M. Vences,et al.  First Large-Scale DNA Barcoding Assessment of Reptiles in the Biodiversity Hotspot of Madagascar, Based on Newly Designed COI Primers , 2012, PloS one.

[15]  Qin Chen,et al.  COI is better than 16S rRNA for DNA barcoding Asiatic salamanders (Amphibia: Caudata: Hynobiidae) , 2012, Molecular ecology resources.

[16]  A. Hausmann,et al.  Barcoding Fauna Bavarica: 78% of the Neuropterida Fauna Barcoded! , 2014, PloS one.

[17]  M. Veith,et al.  Fossorial but widespread: the phylogeography of the common spadefoot toad (Pelobates fuscus), and the role of the Po Valley as a major source of genetic variability , 2007, Molecular ecology.

[18]  C. Wiuf,et al.  Monitoring endangered freshwater biodiversity using environmental DNA. , 2012, Molecular ecology.

[19]  Arie van der Meijden,et al.  Comparative performance of the 16S rRNA gene in DNA barcoding of amphibians , 2005, Frontiers in Zoology.

[20]  R. Jooris,et al.  Potential impact of genome exclusion by alien species in the hybridogenetic water frogs (Pelophylax esculentus complex) , 2009, Biological Invasions.

[21]  V. Helfer,et al.  Phylogeography and Pleistocene refugia of the adder (Vipera berus) as inferred from mitochondrial DNA sequence data , 2006, Molecular ecology.

[22]  M. Veith,et al.  Cryptic niche conservatism among evolutionary lineages of an invasive lizard. , 2012 .

[23]  F. Glaw,et al.  Composition of twenty Green Frog populations ( Pelophylax ) across Bavaria , Germany , 2013 .

[24]  Kevin McDonough.  Amphibian Species of the World: An Online Reference (Version 6) , 2014 .

[25]  J. Plötner,et al.  Morphometric and DNA investigations into European water frogs (Rana kl. esculenta Synklepton (Anura, Ranidae)) from different population systems , 2009 .

[26]  T. Beebee,et al.  The amphibian decline crisis: A watershed for conservation biology? , 2005 .

[27]  Rhys A. Farrer,et al.  Recent introduction of a chytrid fungus endangers Western Palearctic salamanders , 2014, Science.

[28]  Phylogeographic patterns of genetic diversity in the common spadefoot toad, Pelobates fuscus (Anura: Pelobatidae), reveals evolutionary history, postglacial range expansion and secondary contact , 2013, Organisms Diversity & Evolution.

[29]  A. Lambert,et al.  ABGD, Automatic Barcode Gap Discovery for primary species delimitation , 2012, Molecular ecology.

[30]  R. T. Brumfield,et al.  Applications of next-generation sequencing to phylogeography and phylogenetics. , 2013, Molecular phylogenetics and evolution.

[31]  C. Moritz,et al.  DNA barcoding will often fail to discover new animal species over broad parameter space. , 2006, Systematic biology.

[32]  S. Rykena.  Kreuzungsexperimente zur Prüfung der Artgrenzen im Genus Lacerta sensu stricto , 1991 .

[33]  H. Macgregor,et al.  An integrative analysis of phylogenetic relationships among newts of the genus Triturus (family Salamandridae), using comparative biochemistry, cytogenetics and reproductive interactions , 1990 .

[34]  A. Hausmann,et al.  DNA Barcoding the Geometrid Fauna of Bavaria (Lepidoptera): Successes, Surprises, and Questions , 2011, PloS one.

[35]  Do introduced wall lizards (Podarcis muralis) cause niche shifts in a native sand lizard (Lacerta agilis) population? A case study from south-western Germany , 2013 .

[36]  Natalia Ivanova,et al.  Universal primer cocktails for fish DNA barcoding , 2007 .

[37]  D. Wake,et al.  Evolution of mitochondrial relationships and biogeography of Palearctic green toads (Bufo viridis subgroup) with insights in their genomic plasticity. , 2006, Molecular phylogenetics and evolution.

[38]  M. Vences,et al.  DNA barcoding amphibians and reptiles. , 2012, Methods in molecular biology.

[39]  P. Hebert,et al.  DNA barcoding largely supports 250 years of classical taxonomy: identifications for Central European bees (Hymenoptera, Apoidea partim) , 2015, Molecular ecology resources.

[40]  D. Baird,et al.  Environmental Barcoding: A Next-Generation Sequencing Approach for Biomonitoring Applications Using River Benthos , 2011, PloS one.

[41]  T. Beebee,et al.  Lusitania revisited: a phylogeographic analysis of the natterjack toad Bufo calamita across its entire biogeographical range. , 2006, Molecular phylogenetics and evolution.

[42]  R. Murphy,et al.  Universal COI primers for DNA barcoding amphibians , 2012, Molecular ecology resources.

[43]  H. Mooney,et al.  Invasive Alien Species in an Era of Globalization , 2007 .

[44]  P. Hebert,et al.  DNA barcoding: how it complements taxonomy, molecular phylogenetics and population genetics. , 2007, Trends in genetics : TIG.

[45]  U. Schulte,et al.  Amphibien und Reptilien im anthropogenen Klimawandel: Was wissen wir und was erwarten wir? , 2010 .

[46]  M. Vences,et al.  Cold Code: the global initiative to DNA barcode amphibians and nonavian reptiles , 2013 .

[47]  M. Vences,et al.  DNA barcoding Madagascar's amphibian fauna , 2014 .

[48]  M. Vences,et al.  Deciphering amphibian diversity through DNA barcoding: chances and challenges , 2005, Philosophical Transactions of the Royal Society B: Biological Sciences.

[49]  N. Gemmell,et al.  Underestimation of Species Richness in Neotropical Frogs Revealed by mtDNA Analyses , 2007, PloS one.

[50]  C. Meyer,et al.  DNA Barcoding: Error Rates Based on Comprehensive Sampling , 2005, PLoS biology.

[51]  Z. Nagy,et al.  Phylogeography of western Palaearctic reptiles - Spatial and temporal speciation patterns $ , 2007 .

[52]  Robert C. Edgar,et al.  MUSCLE: multiple sequence alignment with high accuracy and high throughput. , 2004, Nucleic acids research.

[53]  P. Lymberakis,et al.  Slow worm, Anguis fragilis (Reptilia: Anguidae) as a species complex: Genetic structure reveals deep divergences. , 2010, Molecular phylogenetics and evolution.

[54]  M. Veith,et al.  Rapid genetic assimilation of native wall lizard populations (Podarcis muralis) through extensive hybridization with introduced lineages , 2012, Molecular ecology.

[55]  G. L. Buffon,et al.  Histoire naturelle, générale et particulière, des reptiles : , 1802 .

[56]  M. Araújo.  The coincidence of people and biodiversity in Europe , 2003 .

[57]  V. Savolainen,et al.  Towards writing the encyclopaedia of life: an introduction to DNA barcoding , 2005, Philosophical Transactions of the Royal Society B: Biological Sciences.

[58]  Carl von Linné.  Systema Naturae: Per Regna Tria Naturae, Secundum Classes, Ordines, Genera, Species, Cum Characteribus, Differentiis, Synonymis, Locis, , 2011 .

[59]  D. Janzen,et al.  DNA barcodes distinguish species of tropical Lepidoptera. , 2006, Proceedings of the National Academy of Sciences of the United States of America.

[60]  C. K. Dodd,et al.  Challenges in Evaluating the Impact of the Trade in Amphibians and Reptiles on Wild Populations , 2005 .

[61]  M. Araújo,et al.  Climate warming and the decline of amphibians and reptiles in Europe , 2006 .

[62]  P. Beerli,et al.  Widespread unidirectional transfer of mitochondrial DNA: a case in western Palaearctic water frogs , 2008, Journal of evolutionary biology.

[63]  R. Sacchi,et al.  An ancient lineage of slow worms, genus Anguis (Squamata: Anguidae), survived in the Italian Peninsula. , 2013, Molecular phylogenetics and evolution.

[64]  C. Meyer.  Molecular systematics of cowries (Gastropoda: Cypraeidae) and diversification patterns in the tropics , 2003 .

[65]  João F. Gonçalves,et al.  Updated distribution and biogeography of amphibians and reptiles of Europe , 2014 .

[66]  W. Ulrich,et al.  From southern refugia to the northern range margin: genetic population structure of the common wall lizard, Podarcis muralis , 2013 .

[67]  M. Valvo,et al.  Variation of Sicilian pond turtles, Emys trinacris - What makes a species cryptic? , 2006 .

[68]  A. Freitas,et al.  New evidence on the systematic and phylogenetic position of Parides burchellanus (Lepidoptera: Papilionidae) , 2008, Molecular ecology resources.

[69]  Litvinchuk,et al.  Cryptic speciation in Pelobates fuscus (Anura, Pelobatidae): evidence from DNA flow cytometry , 2001 .